Retinal cell transplant

ABSTRACT

Surgical instruments, surgical techniques, grafts, cell and tissue isolation techniques, and a method for transplanting retinal cells, including photoreceptors, retinal pigment epithelium, and choroidea within their normal configuration, or all three tissues in a co-transplantation procedure are provided.

This application is a continuation of Ser. No. 07/566,996 filed Aug. 13,1990, abandoned, which is a continuation in part of Ser. No. 07/394,377filed Aug. 14, 1989, abandoned.

BACKGROUND OF THE INVENTION

The present invention relates in general to surgical instruments,surgical techniques, and cell and tissue isolation techniques. Moreparticularly, the present invention is directed to a surgical tool fortransplanting retinal cells, epithelium and choroidea within theirnormal planar configuration, a graft for transplantation to thesubretinal region of the eye, a method for preparing such grafts fortransplantation, and a method for reconstructing dystrophic retinas,retinal pigment epithelial layers and choroids.

The retina is the sensory epithelial surface that lines the posterioraspect of the eye, receives the image formed by the lens, transducesthis image into neural impulses and conveys this information to thebrain by the optic nerve. The retina comprises a number of layers,namely, the ganglion cell layer, inner plexiform layer, inner nuclearlayer, outer plexiform layer, outer nuclear layer, photoreceptor innersegments and outer segments. The outer nuclear layer comprises the cellbodies of the photoreceptor cells with the inner and outer segmentsbeing extensions of the cell bodies.

The choroid is a vascular membrane containing large branched pigmentcells that lies between the retina and the sclerotic coat of thevertebrate eye. Immediately between the choroid and the retina is theretinal pigment epithelium which forms an intimate structural andfunctional relationship with the photoreceptor cells.

Several forms of blindness are primarily related to the loss ofphotoreceptor cells caused by defects in the retina, retinal pigmentepithelium, choroid or possibly other factors (e.g. intense light,retinal detachment, intraocular bleeding). In several retinaldegenerative diseases select populations of cells are lost.Specifically, in macular degeneration and retinitis pigmentosa retinalphotoreceptors degenerate while other cells in the retina as well as theretina's central connections are maintained. In an effort to recoverwhat was previously thought to be an irreparably injured retina,researchers have suggested various forms of grafts and transplantationtechniques, none of which constitute an effective manner forreconstructing a dystrophic retina.

The transplantation of retinal cells to the eye can be traced to areport by Royo et al., Growth 23: 313-336 (1959) in which embryonicretina was transplanted to the anterior chamber of the maternal eye. Avariety of cells were reported to survive, including photoreceptors.Subsequently del Cerro was able to repeat and extend these experiments(del Cerro et al., Invest. Ophthalmol. Vis. Sci. 26: 1182-1185, 1985).Soon afterward Turner, et al. Dev. Brain Res. 26:91-104 (1986) showedthat neonatal retinal tissue could be transplanted into retinal wounds.

In related studies, Simmons et al., Soc. Neurosci. Abstr. 10: 668 (1984)demonstrated that embryonic retina could be transplanted intracranially,survive, show considerable normal development, be able to innervatecentral structures, and activate these structures in a light-dependentfashion. Furthermore, these intracranial transplants could elicitlight-dependent behavioral responses (pupillary reflex) that weremediated through the host's nervous system. Klassen et al., Exp. Neurol.102: 102-108 (1988) and Klassen et al. Proc. Natl. Acad., Sci. USA84:6958-6960 (1987).

Li and Turner, Exp. Eye Res. 47:911 (1988) have proposed thetransplantation of retinal pigment epithelium (RPE) into the subretinalspace as a therapeutic approach in the RCS dystrophic rat to replacedefective mutant RPE cells with their healthy wild-type counterparts.According to their approach, RPE were isolated from 6- to 8-day oldblack eyed rats and grafted into the subretinal space by using a lesionparadigm which penetrates through the sclera and choroid. A 1 μlinjection of RPE (40,000-60,000 cells) was made at the incision siteinto the subretinal space by means of a 10 μl syringe to which wasattached a 30 gauge needle. However, this method destroys the cellularpolarity and native organization of the donor retinal pigment epitheliumwhich is desirable for transplants.

del Cerro, (del Cerro et al., Invest. Ophthalmol. Vis. Sci. 26:1182-1185, 1985) reported a method for the transplantation of tissuestrips into the anterior chamber or into the host retina. The stripswere prepared by excising the neural retina from the donor eye. Theretina was then cut into suitable tissue strips which were then injectedinto the appropriate location by means of a 30 gauge needle ormicropipette with the width of the strip limited to the inner diameterof the needle (250 micrometers) and the length of the strip being lessthan 1 millimeter. While del Cerro reports that the intraoculartransplantation of retinal strips can survive, he notes that theprocedure has some definite limitations. For instance, his techniques donot allow for the replacement of just the missing cells (e.g.photoreceptors) but always include a mixture of retinal cells. Thus,with such a transplant appropriate reconstruction of the dystrophicretina that lacks a specific population of cells (e.g., photoreceptors)is not possible.

del Cerro et al., Neurosci. Lett. 92: 21-26, 1988, also reported aprocedure for the transplantation of dissociated neuroretinal cells. Inthis procedure, the donor retina is cut into small pieces, incubated intrypsin for 15 minutes, and triturated ii nto a single cell suspensionby aspirating it through a fine pulled pipette. Comparable to the Li andTurner approach discussed above, this procedure destroys the organizednative structure of the transplant, including the donor outer nuclearlayer; the strict organization of the photoreceptors with the outersegments directed toward the pigment epithelium and the synapticterminals facing the outer plexiform layer are lost. Furthermore, nomeans of isolating and purifying any given population of retinal cells(e.g. photoreceptors) from other retinal cells was demonstrated.

It is believed by the present inventor that it is necessary to maintainthe photoreceptors in an organized outer nuclear layer structure inorder to restore a reasonable degree of vision. This conclusion is basedon the well known optical characteristics of photoreceptors (outersegments act as light guides) and clinical evidence showing that foldsor similar, even minor disruptions in the retinal geometry can severelydegrade visual acuity.

SUMMARY OF THE INVENTION

Among the objects of the present invention, therefore, may be noted theprovision of a method for preparation of a graft for use in thereconstruction of a dystrophic retina; the provision of such a methodwhich conserves relatively large expanses of the tissue harvested from adonor eye; the provision of such a method in which the polarity andorganization of the cells at the time of harvest are maintained in thegraft; the provision of a graft for use in the reconstruction of adystrophic retina; the provision of such a graft which facilitatesregrowth of photoreceptor axons by maintaining the polar organization ofthe photoreceptor and the close proximity of their postsynaptic targetswith the adjacent outer plexiform layer upon transplantation; theprovision of a surgical tool for use in the transplantation method whichallows appropriate retinotopic positioning and which protectsphotoreceptors or other grafted tissue from damage prior to and as thesurgical device is positioned in the eye; and the provision of a methodfor transplantation of grafts to the subretinal area of an eye.

Briefly, therefore, the present invention is directed to a method forthe preparation of a graft for transplantation to the subretinal area ofa host eye. The method comprises providing donor tissue and harvestingfrom that tissue a population of cells selected from retinal cells,epithelial tissue or choroidal tissue, the population of cells havingthe same organization and cellular polarity as is present in normaltissue of that type. The population of cells is laminated to a non-toxicand flexible composition which substantially dissolves at bodytemperature.

The present invention is further directed to a method for thepreparation of a graft comprising photoreceptor cell bodies fortransplantation to the subretinal area of a host eye. The methodcomprises providing a donor retina containing a layer of photoreceptorcell bodies. The layer of photoreceptor cell bodies is isolated from atleast one other layer of cells of the donor retina in a manner thatmaintains the layer of photoreceptor cell bodies in the sameorganization and cellular polarity as is present in normal tissue ofthat type.

The present invention is further directed to a graft for transplantationto the subretinal area of a host eye. The graft comprises a laminate ofa non-toxic and flexible composition which substantially dissolves atbody temperature and a population of cells harvested from a donor eye,the population of cells being selected from retinal cells, epithelialtissue and choroidal tissue. The population of cells has the sameorganization and cellular polarity as is present in normal tissue ofthat type.

The present invention is further directed to a graft for transplantationto the subretinal area of a host eye. The graft comprises a populationof photoreceptor cell bodies harvested from a donor retina, thepopulation of photoreceptor cell bodies having the same organization andcellular polarity as is present in normal tissue of that type, the grafthaving an essential absence of at least one layer of cells present inthe donor retina.

The present invention is further directed to a method for transplantingto the subretinal area of a host's eye a graft comprising a populationof cells. The method comprises providing a graft comprising a populationof cells selected from retinal cells isolated from at least one otherlayer of cells within the retina, epithelial tissue and choroidaltissue, the population of cells being maintained in the sameorganization an t cellular polarity as is present in normal tissue ofthat type. An incision is made through the host's eye, the retina is atleast partially detached to permit access to the subretinal area and thegraft is positioned in the accessed subretinal area.

The present invention is further directed to an instrument for theimplantation of an intact planar cellular structure between the retinaand supporting tissues in an eye. The instrument comprises an elongatesupporting platform for holding the planar cellular structure. Theplatform has a distal end for insertion into an eye, and a proximal end.The distal edge of the platform is convexly curved for facilitating theinsertion of the platform into the eye and the advancement of theplatform between the retina and the supporting tissues. The instrumenthas a side rail on each side of the platform for retaining the planarcellular structure on the platform, the distal ends of the side railsbeing rounded, and the distal portions of the side rails tapering towardthe distal end of the platform to facilitate the insertion of theinstrument between the retina and the supporting tissue.

The present invention is further directed to an instrument for theimplantation of an intact planar cellular structure between the retinaand supporting tissues in an eye. The instrument comprises an elongatetube, having a flat, wide cross-section, with a top, a bottom forsupporting the planar cellular structure, and opposing sides. The tubehas a beveled distal edge facilitating the insertion of the tube intothe eye and the advancement of the tube between the retina and thesupporting tissues. The instrument also comprises plunger means forejecting a planar cellular structure from the distal end of the tube.

The present invention is further directed to a kit for transplantationof a graft to the subretinal area of a host eye. The kit contains agraft comprising a population of cells selected from retinal cells,epithelial tissue and choroidal tissue, the population of cells beingmaintained in the same organization and cellular polarity as is presentin normal tissue of that type. The kit additionally contains a surgicalinstrument comprising an elongate tube, having a flat, widecross-section, with a top, a bottom for supporting the planar cellularstructure, and opposing sides. The tube has a beveled distal edgefacilitating the insertion of the tube into the eye and the advancementof the tube between the retina and the supporting tissues. The surgicalinstrument also comprises plunger means for ejecting a planar cellularstructure from the distal end of the tube.

Other objects and features of the invention will be in part apparent andin part pointed out hereinafter.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a photograph of a cryostat section of normal rat retina as setforth in Example 1;

FIG. 2 is a photograph of a blinded rat retina following constantillumination as set forth in Example 1;

FIG. 3 is a schematic of a donor retina;

FIG. 4 is a schematic of a flattened retina;

FIG. 5 is a schematic of a flattened retina mounted to a substrate;

FIG. 6 is a schematic of a sectioned retina mounted to a substrate;

FIG. 7 is a schematic of a laminate comprising a retina section on asupporting, stabilizing substrate;

FIG. 8 is a schematic top plan view of the laminate of FIG. 7, showing agraft (dashed lines) comprising a photoreceptor cell layer and asupporting, stabilizing substrate;

FIG. 9 is a perspective view of a first embodiment of an instrumentadapted for implanting an intact planar cellular structure between theretina and supporting tissues in an eye;

FIG. 10 is a side elevation view of a second embodiment of an instrumentadapted for implanting an intact planar cellular structure between theretina and supporting tissues in an eye, with the plunger in itsretracted position, and with portions broken away to show detail;

FIG. 11 is a top plan view of the instrument shown in FIG. 10;

FIG. 12 is a side elevation view of the instrument of the secondembodiment, with the plunger in its extended position, and with portionsbroken away to show detail;

FIG. 13 is a top plan view of the instrument shown in FIG. 12;

FIG. 14 is a partial longitudinal cross-sectional view of theinstrument, showing part of a planar cellular structure loaded therein;

FIG. 15 is a top plan view of a first alternative construction of thesecond embodiment;

FIG. 16 is a top plan view of a second alternative construction of thesecond embodiment;

FIG. 17 is a top plan view of a third alternative construction of thesecond embodiment; and

FIG. 18 is a top plan view of the second embodiment, showing analternative plunger means.

FIG. 19 is a horizontal section through an eye illustrating atrans-corneal surgical approach;

FIG. 20 is a horizontal section through an eye illustrating atrans-choroidal and scleral surgical approach;

FIG. 21 is a photograph of transplanted photoreceptors as set forth inExample 1;

FIG. 22 is a photograph of donor photoreceptors transplanted at theposterior pole of the recipient eye as set forth in Example 1;

FIG. 23 shows the interface between the transplant and the adjacentretina devoid of outer nuclear layer as set forth in Example 1;

FIG. 24 is a photograph illustrating FITC fluorescent micrograph ofantibody Ret P-1 specific for opsin as set forth in Example 1;

FIG. 25 is a series of photograph panels illustrating in A, transplantedphotoreceptors attached to recipient or host retina, in B, fluorescentmicrograph showing transplanted cells showing DiI fluorescence,identifying them as transplanted tissue, and in C, a micrographillustrating FITC fluorescence of antibody specific for opsin as setforth in Example 1;

FIG. 26 comprises two micrograph panels, in A, transplant of nature ratphotoreceptors to adult light damaged recipient or host, and in B,transplant of human photoreceptor from adult donor to adult lightdamaged rat host or recipient as set forth in Example 4;

FIGS. 27a, 27b, 27c, 27d are ¹⁴ C 2-deoxyglucose (2DG) autoradiographs,DYST-dystrophic, TRANS-transplant as set forth in Example 5; and

FIGS. 28a, 28b, 28c and 28d are a is series of photographs showingpupillary reflex to light as set forth in Example 9.

DETAILED DESCRIPTION

As used herein, the term "donor" shall mean the same or differentorganism relative to the host and the term "donor tissue" shall meantissue harvested from the same or different organism relative to thehost.

Several forms of blindness such as retinitis pigmentosa, retinaldetachment, macular degeneration, and light exposure-related blindness,are primarily related to the loss of the photoreceptors in the eye.However, destruction of the photoreceptors does not necessarily lead tothe loss of the remaining retina or axons that connect the retina to thebrain. Surprisingly, it has been discovered that some degree of visioncan be restored by replacing damaged photoreceptors with photoreceptorsharvested from a donor and which are maintained in their originalorganization and cellular polarity.

FIG. 1 is a photograph of a cryostat section of normal rat retina. FIG.2 is a photograph of a cryostat section of a rat retina followingconstant illumination which destroys the photoreceptor (outer nuclear)layer while leaving other retinal layers and cells largely intact. Inthese and subsequent figures, the retina or layers thereof, e.g., theganglion cell layer ("G"), inner plexiform layer ("IPL"), inner nuclearlayer ("INL"), outer plexiform layer ("OPL"), outer nuclear layer("ONL"), inner segments ("IS"), outer segments ("OS"), and retinalpigment epithelium ("RPE"), are shown, respectively, from top to bottom.

Referring now to FIG. 3, a graft comprising photoreceptor cells isprepared in accordance with a method of the present invention byremoving a donor retina 50 comprising inner retina layers 52 andphotoreceptor layer 54 from a donor eye. The donor retina 50 isflattened (FIG. 4) by making a plurality of cuts through the retina fromlocations near the center of the retina to the outer edges thereof (seeFIG. 8). Cuts can be made in other directions if necessary.

As shown in FIG. 5, the flattened retina 56 is placed with thephotoreceptor side 54 down on a gelatin slab 58 which has been surfacedso as to provide a flat surface 60 that is parallel to the blade of avibratome apparatus. The gelatin slab 58 is secured to a conventionalvibratome chuck of the vibratome apparatus. Molten four to five per centgelatin solution is deposited adjacent the flattened retina/gelatinsurface interface 61 and is drawn by capillary action under theflattened retina causing the flattened retina to float upon the gelatinslab 58. Excess molten gelatin is promptly removed and the floatingflattened retina is then cooled to approximately 4° C. with ice-coldRinger's solution that surrounds the gelatin block to cause the moltengelatin to gel and hereby coat the bottom surface of the flattenedretina and adhere it to the gelatin block.

As shown in FIG. 6, the inner retina portion 52 is sectioned from thetop down at approximately 20 to 50 millimicrons until the photoreceptorlayer 54 is reached, thereby isolating the photoreceptor layer from theinner layers of the retina, i.e., the ganglion cell layer, innerplexiform layer, inner nuclear layer, and outer plexiform layer. Whenthe photoreceptor layer is reached, the vibratome stage is advanced anda section from approximately 200 to 300 millimicrons thick obtained asshown in FIG. 7. The thickness of this section is sufficient to undercutthe photoreceptor and form a laminate 62 consisting of a layer ofphotoreceptor cells and the gelatin adhered thereto.

As shown in FIG. 8, the laminate 62 is cut vertically along the dashedlines to create a graft 63 having a size appropriate fortransplantation. The surface of the graft should have a surface areagreater than about 1 square millimeter, preferably greater than 2 squaremillimeters, and most preferably greater than 4 square millimeters or aslarge as may be practically handled. Thus constructed, the graft maysubtend a considerable extent of the retinal surface.

The gelatin substrate adds mechanical strength and stability to theeasily damaged photoreceptor layer. As a result, the flattened retinaltissue is less likely to be damaged and is more easily manipulatedduring the transplantation procedure.

Gelatin is presently preferred as a substrate because of itsflexibility, apparent lack of toxicity to neural tissue and ability todissolve at body temperature.

However, other compositions such as ager or agarose which also have thedesirable characteristics of gelatin may be substituted. Significantly,gelatin has not been found to interfere with tissue growth orpost-transplant interaction between the graft and the underlying retinalpigment epithelium.

Gelatin is presently preferred as an adhesive to laminate the retinaltissue to the substrate. However, other compositions, including lectinssuch as concanavalin A, wheat germ agglutin, or photoreactive reagentswhich gel or solidify upon exposure to light and which also have thedesirable characteristics of gelatin may be substituted.

Advantageously, the gelatin or other substrate may additionally serve asa carrier for any of a number of trophic factors such as fibroblastgrowth factor, pharmacologic agents including immunosuppressants such ascyclosporin A, anti-inflamation agents such as dexamethasone,anti-angiogenic factors, anti-glial agents, and anti-mitotic factors.Upon dissolution of the substrate, the factor or agent becomes availableto impart the desired effect upon the surrounding tissue. The dosage canbe determined by established experimental techniques. The substrate maycontain biodegradable polymers to act as slow release agents forpharmacologic substances that may be included in the substrate.

The thickness of the graft comprising the sectioned flattened retinaltissue and the substrate as discussed above is only approximate and willvary as donor material varies. In addition, sectioning may befacilitated and vibratome thickness further calibrated from histologicalmeasurements of the thickness of the retina, thereby providing furtherguides to sectioning depth. Appropriate sectioning thicknesses or depthmay be further determined by microscopic examination and observation ofthe sections.

As an alternative to mechanical, e.g., microtome, sectioning, the donorretina may be chemically sectioned. Specifically it is known thatneurotoxic agents such as kainic acid are toxic to cells in all retinallayers except the photoreceptor layer (i.e., kainic acid does not damagephotoreceptor cells). Therefore, if the donor retina is treated with anappropriate neurotoxic agent, the photoreceptor layer can be isolated.This technique has the advantage of maintaining the retinal MUller cells(which are not killed by kainic treatment) with the photoreceptor cells.Since it is known that MUller cells help maintain photoreceptor cells(both biochemically and structurally), the isolation of MUller cellsalong with the photoreceptor cells could be advantageous.

If desired, the graft may additionally contain the retinal pigmentepithelial cells. Because the RPE is tenuously adherent to the retina,mechanical detachment of the retina from a donor eye ordinarily willcause the RPE to separate from the retina and remain attached to thechoroid. However, through the use of enzymatic techniques such as thosedescribed in Mayerson et al., Invest. Opthalmol. Vis, Sci. 25:1599-1609, 1985, the retina can be separated from the donor eye with theRPE attached. In accordance with the present invention, graftscomprising the choroid may additionally be prepared. To do so, thechoroid is stripped off of the scleral lining of the eye (with orwithout the RPE attached), and flattened by making radial cuts. Thedonor choroid may then be adhered to a substrate as previously describedfor the photoreceptor cells and/or combined with a photoreceptor layerwhich has been prepared as described above to form a laminate comprisinga photoreceptor layer adhered to a substrate, a RPE layer and achoroidal layer.

Referring again to the Figures there is shown preferred embodiments forthe surgical instruments of this invention. The surgical instruments aredescribed in connection with a photoreceptor isolation andtransplantation method. The surgical instruments and methods of thisinvention are particularly adapted for isolation and transplantation ofan intact sheet of cells from a donor retina to a recipient retina andare characterized by the maintenance of cell organization of thetransplanted tissue layer.

A first embodiment of an instrument for implanting an intact planarcellular structure between the retina and supporting tissues in an eyeis indicated generally as 10 in FIG. 9. The instrument 10 may be madefrom acrylic, or some other suitable material that is flexible andsterilizable. The instrument 10 comprises an elongate platform 11 forholding the planar cellular structure. The platform 11 has a distal end16 for insertion into the eye of the recipient, and a proximal end 18.As shown and described herein the platform 11 is approximately 2 to 10centimeters long, which is an appropriate length for making implants inrodents and lower primates. The platform 11 must be sufficiently long toextend into the eye, between the retina and the supporting tissue, andthus different platform lengths may be used, depending upon theprocedure being employed and upon the recipient. As shown and describedherein the platform is approximately 2.5 millimeters wide, which issufficiently wide for making implants in rodents and lower primates. Theplatform 11 must be sufficiently wide to carry and intact cellularstructure for implanting, and thus different platform widths may beused, depending upon the recipient.

As shown in FIG. 9, the edge 11a of the platform 11 at the distal end 16is preferably convexly curved to facilitate both the insertion of theinstrument 10 into the eye, and the advancement of the instrumentbetween the retina and the supporting tissue to temporarily detach theretina, with a minimum of trauma. The platform 11 is preferablyconcavely curved (with respect to the top surface of the platform 11)along its longitudinal axis from the distal end 16 to the proximal end18. The curvature of the platform 11 facilitates the manipulation of theinstrument 10 within the eye, particularly the manipulation of theinstrument between the retina and the supporting tissue on the curvedwalls of the eye. The radius of the curvature of the platform 11 willdepend upon the procedure and the recipient.

The platform 11 has side rails 12 and 14 on opposite sides for retainingthe planar cellular structure on the platform. As shown in FIG. 9, thedistal portions 12a and 14a of the side rails taper from a pointintermediate the distal and proximal ends of the side rails toward theirdistal ends. The distal ends of the side rails terminate in smoothlycurved ends 12b and 14b, which are proximal of the distal end 16 of theplatform. The offset of the distal ends of the rails, together withtheir rounded configuration facilitates the insertion of the instrumentinto the eye and the advancement of the instrument between the retinaand the supporting tissue. As shown and described herein, the proximalportions of the side rails 12 and 14 are approximately 1 millimeterhigh, while the distal portions 12a and 14a taper to about 0.5millimeters. The height of the side rails is made as small as possible,but they must be slightly greater than the thickness of the planar cellstructure and the supporting substrate, and thus may vary depending onthe donor and the type of implantation being made (ie, how many celllayers are being implanted and thickness of the substrate).

A second embodiment of an instrument for implanting an intact planarcellular structure between the retina and supporting tissues in an eyeis indicated generally as 30 in FIGS. 10-14, and 18. The instrument 30may be made from polyethylene, or some other suitable material that isflexible and sterilizable. For example, the instrument might be made ofsilicone rubber or silastic. The instrument 30 comprises an elongatetube 32 having a flat, wide cross-section, with a top 32a, a bottom 32bfor supporting the planar cellular structure, and opposing sides 32c and32d. The tube 32 has a distal end 34 for insertion into the eye, and aproximal end 36. The distal end 34 of the tube 32 is open for thedischarge of the planar cellular structure. The instrument 30 of thesecond embodiment is preferable to the instrument 10 of the firstembodiment in at least one respect because the tube 32 has a top 32awhich provides better protection for the planar cellular structure to beimplanted than the open platform 11.

As shown and described herein the tube 32 is approximately 3.5centimeters long, which is an appropriate length for making implants inrodents and lower primates. The tube 32 must be sufficiently long toextend into the eye, between the retina and the supporting tissue, andthus the different tube lengths may be used, depending upon theprocedure being employed and upon the recipient. As shown and describedherein the tube is approximately 2.5 centimeters wide, which issufficiently wide for making implants in rodents and lower primates. Thetube must be sufficiently wide to carry an intact cellular structure forimplanting, and thus different tube widths may be used, depending uponthe recipient. As shown and described herein, the sides 32c and 32d areapproximately 0.75 millimeters high. The height of the sides is made assmall as possible, but they must be slightly greater than the thicknessof the planar cell structure and substrate, and thus may vary dependingon the donor and the type of implantation being made (i.e. how many celllayers are being implanted and thickness of the substrate).

The distal end 34 of the tube can be beveled to facilitate both theinsertion of the tube into the eye, and the advancement of the tubebetween the retina and the supporting tissues, with a minimum of trauma.The end is preferably beveled at about 45°, from the top 32a to thebottom 32b. As shown in FIGS. 10 and 12, the distal end 34 of the tube32 is also preferably raked transversely across the tube (i.e. from side32c to 32d) toward the proximal end. The rake angle is preferably about45°. The raked distal end also facilitates the insertion of the tubeinto the eye, and the advancement of the tube between the retina and thesupporting tissue. Moreover, raking the distal end eliminates a sharpcorner that could damage tissue.

The tube 32 is preferably concavely curved along its longitudinal axisfrom the distal end 34 to the proximal end 36, so that the top 32a is onthe inside of the curve, and the bottom 32b is on the outside of thecurve. The curvature of the tube facilitates the manipulation of theinstrument 30 within the eye, particularly the manipulation of theinstrument between the retina and the supporting tissue on the curvedwalls of the eye. The radius of the curvature of the tube will depend onthe procedure and on the recipient.

The instrument 30 also comprises plunger means. As shown in FIGS. 10-14,the plunger means is preferably a flat plunger 40 slidably received inthe tube so that relative sliding motion between the tube 32 and theplunger 40 urges a planar cellular structure that is in the tube out thedistal end of the tube. The plunger 40 may be made ofpolymethylmethacrylate. The proximal end of the plunger 40 projects asufficient amount from the proximal end of the tube 32 that the end ofthe plunger can be manipulated even when the distal portion of the tubeis in an eye. The preferred method of operating the instrument 30 isthat once the distal end of the tube is properly located within thesubretinal area, the plunger 40 is held in place as the tube 32 isgradually withdrawn to eject the cellular structure.

Alternatively, as shown in FIG. 18, the plunger means may comprise meansfor applying hydraulic pressure on the contents of the tube. In thiscase the proximal end 36 of the tube 32 is connected to a line 41connected to a source of fluid under pressure. Fluid can be selectivelysupplied via the line 41 to the proximal end of the tube, to displacethe contents of the tube. The fluid may be viscous, for example a 2%carboxymethylcellulose, or non-viscous. Particularly in the later case,it may be desirable to have a block 43 of gelatin or some othersubstance in the tube to act as a mechanical plunger and to separate thefluid from the cell structure being implanted. Gelatin is satisfactorybecause it is a semi-solid, and because it will dissolve harmlessly ifit is ejected from the tube.

As shown in FIGS. 10-13, the instrument 30 preferably also includes alumen 42, extending generally parallel with the tube 32. As used herein,lumen refers to any tube-like vessel, whether separately provided orformed as a passageway in another structure. The lumen 42 is attached toone of the sides of the tube 32, and preferably side 32c so that thedistal end of the tube rakes away from the lumen. The lumen 42 has adistal end 44 generally adjacent the distal end of the tube, andpreferably slightly advanced relative to the distal end of the tube. Theproximal end 46 is remote from the distal end, and may be provided witha connector 48 for connection with a source of fluid under pressure.Thus the lumen 42 can eject a stream of fluid from its distal end 44which creates a fluid space ahead of the instrument, which helpsseparate or detach the retina from the supporting tissue as theinstrument is advanced. The fluid may be a saline solution, or someother fluid that will not harm the delicate eye tissues. Varioussubstances, such as anti-oxidants, anti-inflammatories, anti-mitoticagents and local anesthetics can be provided in the fluid for treatmentof the eye or implanted tissue.

The raked distal end of the tube 32 follows generally in the path openedby the fluid, thus minimizing direct contact of the instrument and theeye tissue. The distal end of the lumen may be beveled to facilitate theadvancement of the instrument, particularly at times when fluid is notbeing ejected from the lumen. The end is preferably beveled at about45°. Of course, rather than provide a separate lumen 42, the lumen couldbe formed integrally in the walls of the tube 32.

A first alternate construction of instrument 30 is indicated as 30A inFIG. 15. The instrument 30A is very similar in construction toinstrument 30, and corresponding parts are identified with correspondingreference numerals. However, unlike instrument 30, the instrument 30Aincludes a fiber optic filament 64 extending generally parallel withlumen 42, and positioned between the lumen 42 and the tube 32. The fiberoptic filament 64 facilitates the manipulation of the instrument and theproper placement of the implant in two ways: a light source can beprovided at the proximal end of the fiber optic filament so that thefilament provides light at the distal end of the instrument, tofacilitate the visual observation procedure through the pupil.Alternatively, a lens could be provided at the proximal end of the fiberoptic filament so that the filament can also be used for directobservation at the distal end of the instrument.

Additionally, the fiber optic filament could allow for laser-lightcautery to control subretinal bleeding. Of course, rather than provide aseparate fiber optic filament 64, fiber optic filaments could beincorporated into the walls of the tube 32 or the lumen 42.

A second alternative construction of instrument 30 is indicated as 30Bin FIG. 16. The instrument 30B is very similar in construction toinstrument 30, and corresponding parts are identified with correspondingreference numerals. However, unlike instrument 30, the instrument 30Bincludes a lumen 66 extending generally parallel with lumen 42, andpositioned between the lumen 42 and the tube 32. The lumen 66 allows forthe aspiration of material from the distal end of the instrument. Theproximal end of the lumen 66 can be connected to a source of suction, toremove excess fluid and debris. It is possible to incorporate the lumen66 into the wall of the tube 32.

A third alternative construction of the instrument 30 is indicated as30C in FIG. 17. The instrument 30C is very similar in construction toinstrument 30, and corresponding parts are identified with correspondingreference numerals. However, unlike instrument 30, the instrument 30Cincludes a pair of lead wires 65, terminating in an electrode 67 attheir distal ends. The electrode 67 allows for cauterization of bloodvessels. The proximal ends of the leads 65 can be connected to a sourceof electrical power to seal broken blood vessels. It is possible toincorporate the leads 65 into the wall of the tube 32.

Of course, two or more of the features described with respect to thealternate embodiments 30A, 30B, and 30C could be combined, if desired.

To transplant the retinal cells, including photoreceptors, the host eyeis prepared so as to reduce bleeding and surgical trauma. A transcornealsurgical approach to the subretinal space is one such approach and itwill be understood that other surgical approaches, such as transscleraland choroidal may also be used. The preferred surgical approach in therodent, FIG. 19, includes making a transverse incision 70 in a cornea 72of sufficient size so as to allow insertion of a surgical instrumentillustrated schematically by reference characters 10 or 30. Theinstrument 10 is advanced under the iris, through the cornea 72 and tothe ora serrata 74 as illustrated in FIG. 19. The iris should be dilatedfor example, with topical atropine. When the instrument 10 is used, itdetaches the retina as it is advanced under the retina and into thesub-retinal space to the posterior pole 76 of the eye.

The channel defined by the side rails 12, 14 and the intermediate cellsupporting platform provides for the graft comprising a photoreceptorlayer 54 attached to the gelatin substrate to be placed on theinstrument 10 and guided into the sub-retinal space, preferably withforceps or other suitable instruments. After positioning thephotoreceptor layer at the desired transplant site, the gelatin is heldin position with the forceps while the carrier is removed. The edges ofthe corneal incision are abutted after removal of the forceps to allowrapid, sutureless healing. The eye should be patched during recovery.

If the surgical instrument 30 (FIGS. 10-14, and 18) is used instead ofthe instrument 10, the graft comprising intact generally planar sheet 54of donor photoreceptors attached to the gelatin substrate 62 is drawninto the elongate tube 32. The instrument 30 is then inserted through anappropriate sized incision in the cornea and advanced under the iris.The iris will have been dilated, for example, with topical atropine. Theinstrument 30 is advanced to the ora serrata 74 of the host eye. If theinstrument 30 includes a lumen 42, the retina is detached by the gentleforce of a perfusate such as a saline-like fluid,carboxymethylcellulose, or 1-2% hyluronic acid ejected from the lumen42. Advantageously, the fluid may additionally contain anti-oxidants,anti-inflammation agents, anesthetics or agents that slow the metabolicdemand of the host retina.

If the instrument 30 does not include a lumen 42, the retina is detachedby the walls of the surgical instrument as it is advanced under theretina and into the subretinal space to the posterior pole 76 of theeye. The graft comprising a photoreceptor layer attached to the gelatinsubstrate is then transplanted by moving the tube 32 in a direction awayfrom the eye while keeping the plunger 40 stationary. The plunger 40 iscarefully withdrawn out of the eye and the edges of the corneal incisionare abutted after removal to allow rapid, sutureless healing. Retinalreattachment occurs rapidly and the photoreceptor sheet is held in placein a sandwich-like arrangement between the retina and the underlying eyetissues. The incision may require suturing.

FIG. 20 depicts a trans-choroidal and scleral surgical approach as analternative to the transcorneal approached described above. Except forthe point of entry, the surgical technique is essentially the same asoutlined above. Nevertheless, the transcorneal approach is preferredbecause it has been found to reduce bleeding and surgical trauma.

A further surgical approach is to diathermize in the pars plana regionto eliminate bleeding. The sclera is then incised and the choroidal andepithelial tissue is diathermized. The surgical tool is then insertedthrough the incision, the retina is intercepted at the ora serrata andthe graft is deposited in the subretinal area otherwise as outlinedelsewhere herein.

In yet a further surgical approach, entry is gained through the parsplana area as outlined above and an incision is made in the retinaadjacent to the retinal macula. The surgical tool is then insertedthrough the retinotomy and into the macular area.

It is known that the retina does not necessarily undergo glial scarformation when it is damaged, unlike the adult central nervous system asdisclosed by Bigami et al., Exp. Eye Res. 28:63-69, (1979), and McConnelet al., Brain Res. 241:362-365 (1982). McConnel et al. suggest that thischaracteristic lack of scar tissue contribute to a potential of retinalcells to regrow severed axons within the eye.

In accordance with the present invention, it has recognized thatregrowth of photoreceptor axons may be facilitated by the proximity ofthe post-synaptic targets of the photoreceptor within the adjacent outerplexiform layer. In addition, growth across substantial interveningneural or glial scar tissue is not necessary in order for transplantedphotoreceptors to make appropriate connections with the recipient retinaincluding neural connections.

The following examples illustrates the invention.

EXAMPLE 1

Experimental Animals

Adult albino rats (Sprague-Dawley) were exposed to constant illuminationaveraging 1900 lux for 2 to 4 weeks as described in O'Steen, Exp.Neurol. 27:194 (1970).

As shown in FIG. 1, this exposure destroys most photoreceptors,eliminating cells of the outer nuclear layer but leaving the remainingneural retina intact. Photoreceptors for transplantation were taken from8-day-old normal rats of the same strain that had been maintained undercolony room illumination (10-20 lux) on a 12 hr/12 hr light/dark cycle.Experimental animals were anesthesized with ketamine and sodiumpentobarbital. A preoperative dose of dexamethasone (10 mg/kg IP) wasalso administered.

Photoreceptor Preparation

The retina from the anesthetized 8-day-old rat was removed, flattenedwith radial cuts and placed with the receptor side down on a gelatinslab secured to the vibratome chuck. Molten gelatin (4-5% solution) wasdeposited adjacent the retina at the retina/gelatin interface and thencooled to 4° C. with ice-cold Ringer's solution. The retina wassectioned at 20 to 50μm until the photoreceptor layer was reached. Whenthe photoreceptor layer was reached, the stage was advanced and a thick(200 to 300 μm) section was taken, undercutting the photoreceptor layersecured to the gelatin base.

DiI Labeling

The isolated outer nuclear layer was cultured overnight with 40 μg/ml ofdiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchloratein Earle's MEM containing 10% fetal calf serum, incubated under 95%/5%oxygen/carbon dioxide mixture at room temperature. Labeling techniquesand fluorescent microscopy were otherwise as outlined by Honig et al.,J. Cell. Biol. 103:17, 1986. DiI was removed from sections that were tobe counterstained with FITC-labeled RET-P1 opsin antibody by priorwashing in acetone.

Surgical Procedure

A transverse incision was made in the cornea sufficient to allowinsertion of a surgical instrument 10 that is 2.5 mm wide with siderails 0.5 mm high or surgical instrument 30. The instrument was advancedunder the iris (dilated with topical atropine) to the ora serrata,detaching the retina. The carrier was then advanced under the retinainto the subretinal space to the posterior pole of the eye. Theinstrument allowed a graft comprising a piece of the photoreceptor layerattached to the gelatin substrate (up to 2.5×4 mm) to be guided into theretinal space by fine forceps. The instrument was then removed while thegelatin was held in position by the forceps. Following removal of theforceps, the edges of the corneal incision were abutted to allow rapid,sutureless healing. The eye was patched during recovery and aprophylactic dose of penicillin was administered. Upon removal of thepatch, a verterinary ophthalmic antibiotic ointment was applied.

Transplant recipients were maintained on a 12 hr/12 hr light/dark cyclewith an average light intensity of 50 lux. Following appropriatesurvival times, the animal was overdosed with pentobarbital and perfusedtranscardially with phosphate buffered 3% paraformaldehyde-2%gluteraldehyde solution. Cryostat sections of both the light-blinded eye(control) and the eye receiving the photoreceptor transplant were thencut (20 μm).

Immunohistochemistry

Antibody labeling for opsin was performed on retinas fixed with 3%paraformaldehyde and cryosectioned at 20 μm. Immunohistocheinicalmethods were otherwise as described in Hicks, et al., J. HistochemCytochem 35:1317 (1987). Elimination of the primary antibody eliminatedspecific labeling for opsin.

Cryostat Sections

Cryostat sections made at 4 weeks post-transplantation are shown inFIGS. 22-24. FIG. 22 is a low-power photomicrograph showing the locationof the photoreceptor transplant (between arrowheads) at the posteriorpole of the host eye Bar=0.5 mm. FIG. 23 is a higher-powerphotomicrograph showing the interface between the transplant and theadjacent retina devoid of outer nuclear layer. Arrows indicate theextent of the transplant (T). Arrowheads indicate three possibleresidual photoreceptors that survived constant illumination. Note theirfusiform shape contrasts with the rounder, more normal shape of thetransplanted photoreceptors. H & E stain. Bar=100 μm. FIG. 24 is a FITCfluorescent micrograph of antibody Ret P-1 specific for opsin in asection adjacent to that shown in FIG. 23. Arrows indicate the extent ofthe transplant. Transplanted cells are labeled for opsin indicating theyare photoreceptors. Some nonspecific fluorescence is evident adjacent tothe transplant. Bar=100 μm.

Cryostat sections made at 3 weeks post-transplantation are shown inFIGS. 25A-C. FIG. 25A is a H & E-stained photomicrograph of a transplantand host retina. Note the cell-sparse layer that resembles the outerplexiform layer interposed between the host retina and the transplant.FIG. 25B is a diI fluorescent photomicrograph of adjacent section.Transplanted photoreceptors show diI fluorescence, identifying them asdonor tissue. FIG. 25C is a FITC fluorescent micrograph of antibodyRET-P1 in a section adjacent to that shown in FIG. 25A. Transplantedcells are labeled for opsin, indicating that they are photoreceptors.Bar=50 μm.

Results

By using the transcorneal approach, it was found that the positioning ofthe photoreceptor layer between the host's retina and the adjacentepithelial and choroidal tissue layers of the eye could be accomplishedwhile minimizing the vascular damage and subsequent bleeding into theeye. In addition, it was found that this approach does not appear todisrupt the integrity of the retina, which reattaches to the back of theeye with the transplanted photoreceptors interposed between the retinaand the RPE. As shown in FIGS. 21-24, retinal reattachment appears to befacilitated in the immediate area of the transplant. (The "T" indicatestransplanted sheets of photoreceptor cells). Using this insertionmethod, it was possible to position the photoreceptors at the posteriorpole of the retina (FIG. 22).

To determine the viability of the transplanted photoreceptors, cryostatsections (20 μm) were made from both the blinded eye (control) and theeye receiving the photoreceptor transplant at 2, 4, or 6 weeks aftertransplantation. It was found that the photoreceptors survivedtransplantation at all times tested (36 out of 54 transplants). In mostinstances, the surviving transplant approximated its size at the time oftransplantation. More importantly, there was no apparent reduction intransplant size with longer survival times, suggesting that thetransplants were stable.

As a control, the contralateral eyes that did not receive aphotoreceptor transplant were examined. In these eyes, the retinaspossessed very few residual photoreceptors located adjacent to the outerplexiform layer and the RPE. However, these residual cells were abnormalin their appearance, having flattened, pyknotic cell bodies instead ofthe rounded cell bodies of normal photoreceptors. Furthermore, theresidual photoreceptors did not form an outer nuclear layer composed ofcolumnarly stacked cell bodies, but instead were found in isolation, orat most appear as a single or double layer of cells (see FIG. 23)located mainly in the peripheral retina.

The transplanted cells were easily distinguished from the residualphotoreceptors by a number of parameters. First, they were found indiscrete patches and have the characteristic columnar stackingarrangement of up to about 12 cell bodies that is characteristic ofphotoreceptor cells in the outer nuclear layer of the normal retina.They did not have the flattened appearance of the residual nativephotoreceptors, but instead have the round, nonpyknotic cell bodytypical of normal transplanted cells. Furthermore, the transplantedphotoreceptors can form rosette configurations, a characteristic oftransplanted and cultured retina while residual photoreceptors were notfound in these configurations.

To eliminate the possibility that the surgical procedure in some mannerinduced the regeneration of native photoreceptors, sham operations wereperformed. All procedures were performed as with the photoreceptortransplants except that no photoreceptors were attached to the insertedgelatin slab. While the retina reattached to the back of the eye, in noinstance were patches of photoreceptors found.

To positively identify the photoreceptor patches in experimental animalsas transplanted tissue, the donor outer nuclear layer was labeled withthe fluorescent marker diI prior to the transplantation. As shown inFIG. 25B, the photoreceptor patches were fluorescently labeled while thehost retina did not show diI fluorescence.

To confirm that the transplants consisted of photoreceptors, amonoclonal antibody specific for opsin, RET-P1 was used. As opsin isfound only in photoreceptors, any cell showing labeling for opsin was,therefore, identified as a photoreceptor. As can be seen in FIGS. 24 and25C, the transplanted cells stain intensely for opsin whereas otherretinal cells are unstained. Positive staining for opsin not onlyidentifies these cells as photoreceptors but indicates that these cellsare still capable of producing the protein moiety of visual pigment.Retina adjacent to the region of the transplant shows only a fewisolated photoreceptor cell bodies (FIG. 23) that do not stain for opsin(FIG. 24) suggesting that they are cones. Their lack of opsin staining,as well as their location and appearance in H & E-stained material,confirms that these cells are the host's residual photoreceptors (FIG.23).

Harvesting the photoreceptor layer from the neonatal retina does notappear to disrupt tissue organization. Once transplanted, thephotoreceptor layer maintained its characteristic columnar arrangementof cell bodies for all survival times examined, thus forming a new outernuclear layer within the host's retina. In some cases, strict polaritywas lost and the rosettes were formed. By light microscopy, the newlayer appeared to be attached to the host's outerplexiform layer (FIGS.22 and 25A). This layer normally is the site of synaptic contact betweenthe photoreceptors and the retina.

EXAMPLE 2

The procedures of Example 1 were repeated except as noted. Substitutedfor the Sprague-Dawley rats were the rd mouse and the RCS rat which areafflicted with inherited retinal degeneration. In the rd mouse it isthought that the deficit resides in the photoreceptor whereas in the RCSit is thought that the deficit resides in the pigment epithelium. Inthese animals, almost all photoreceptors are eliminated while theremaining retina is preserved; either the rd mouse or the RCS rat wereblinded by constant illumination as set forth in Example 1. The rd mouseand the RCS respectively received transplants of immature (7-8 day oldmouse or rat) and mature rat photoreceptors.

rd Mouse

The transplantation technique was adapted to the smaller size of themouse eye. This modification allowed sheets of intact outernuclear layerto be transplanted to the subretinal space of the mouse eye. Neonatal (8days old) photoreceptors were transplanted from rd control mice to thesubretinal space of adult rd mice. Survival times were for 2 weeks to 3months. At all survival times, it was found that the transplantedphotoreceptors survived, becoming physically attached to the outerportion of the host retina and stained positive for opsin. In addition,the host retina became reattached to the pigment epithelium.

In the rd mouse almost all photoreceptors are eliminated by day 21. Itwas found that photoreceptors from a non-dystrophic congenic controlmouse can be transplanted to their appropriate site within the adult rdmouse eye that lacks photoreceptors. These transplanted photoreceptorswere found to survive for as long as tested (3 months). This length oftime is significant since photoreceptors of the rd mouse show signs ofdegeneration after about 2 weeks and are almost completely eliminatedafter 3 weeks. The survival of transplanted photoreceptors from congenicnormal donors to the adult rd mouse within the rd mouse supportsfindings that indicate that the deficit within the rd mouse which causesthe degeneration of photoreceptors is endogenous to the rdphotoreceptors themselves.

RCS Rat

Photoreceptors from 7 to 8 day old RCS controls (normal) weretransplanted to the subretinal space in the eye of adult (3 month old)RCS rats. A two month survival period was allowed because in this timeperiod almost all host photoreceptors degenerate in the RCS rat. It wasfound that the grafted photoreceptors survive transplantation to thesubretinal space of the RCS rat and show histotypic as well asimmunological characteristics of normal photoreceptors. In addition, itwas found that transplanted photoreceptors survive within theirhomotopic location in the RCS rat whereas the RCS's own photoreceptorsdo not.

Results

While it has been found that the transplanted photoreceptors survive,produce opsin, and apparently integrate with the recipient retina, theydo not appear completely normal in that the number of outer segments isreduced. However, photoreceptors lacking outer segments are stillcapable of phototransduction as indicated in Pu et al., J. Neorosci.,4:1559-1576, 1984. The relative scarcity of outer segments has also beennoted in retina transplanted to the tecum. These retina have been shownto be functional as indicated in Simon et al., Soc. Neorosci. Abstr.,10:668, (1984).

Conventional reasoning attributes the observed deficiency in outersegments to be the possible consequence of the lack of appropriateapposition of the RPE to the photoreceptors as indicated in LaVail etal., (1971), noted above. However, it has been found that RPE is presentand in apparently normal apposition to the photoreceptors, thus, thescarcity of outer segments here would not appear to be related toinadequate contact between photoreceptor and RPE. The failure of outersegment growth in the presence of photoreceptor apposition to the RPEhas also been seen following retinal reattachment as reported byAnderson et al., Invest. Opthalmol. Vis, Sci. 24:906-926, 1983.

EXAMPLE 3

The procedures of Example 1 were repeated except as noted.

Donor photoreceptors were originally harvested at the earliestontogenetic time in which the photoreceptors could be isolated fromother portions of the retina (7-8 days old) since it is generallybelieved that more embryonic and undifferentiated neural tissue survivestransplantation far better than more mature and differentiated tissue.To determine the effect of developmental age on photoreceptor survivaland ability to integrate with the host retina, photoreceptors weresubsequently transplanted from 8, 9, 12, 15 and 30 day old rats intolight damaged adults. These show progressive development and maturationof the photoreceptors including mature outer segments (at 15 and 30days).

Using the same criteria as in Example 1, it was found that for all agestested the transplants survived for as long as examined (2 months) andintegrated with the host retina. FIG. 26, panel A, is a photograph of atransplant of mature photoreceptors (30 day old donor) to adult lightdamaged host. (T, Transplant). 120X. These observations suggest thatphotoreceptors have characteristics that differ from other neural tissuethat permits them to be transplanted when they are essentially maturewhile other neural tissue must be at a very immature stage forsuccessful transplantation to occur.

EXAMPLE 4

The procedures of Example 1 were repeated except as noted.Photoreceptors were taken from the retina of donated human eyes(obtained from the Missouri Lions and St. Louis Eye Banks) followingcorneal removal. A portion of the retinas were tested for viability bydye exclusion with trypan blue and didansyl cystine staining. Thephotoreceptors excluded dye and appeared to be in good condition. Hostswere adult albino rats (immune-suppressed with cyclosporin A orimmune-competent) exposed to constant illumination.

With immune-suppression successful transplants were seen at all survivaltimes so far examined (one and two weeks; five of nine cases), showingapparent physical integration with the host retina and maintainingmorphological features of the outer nuclear layer as illustrated in FIG.26B which shows a transplant of human photoreceptors from adult donor toadult light damaged rat host. (T, transplant). 120X.

The transplants stained positive for antiopsin antibody RET-P1,identifying the transplanted cells as photoreceptors and furtherindicating that they are still capable of producing visual pigment. Incontrast, transplants to immune-competent hosts showed signs ofrejection within one week of transplantation. Sham operated animalsshowed no repopulation of the host retina with photoreceptors.

The procedures of Example 1 were repeated except as noted.

The 2DG functional mapping technique developed by Sokoloff et al., J.Neurochem. 28:897-916 (1977) allows the measurement of the relativelevels of neural activity for a given stimulus condition. For thisreason, the 2DG technique appeared to be an appropriate method ofassessing the functional characteristics of the transplant and itsability to activate the light damaged retina.

Accordingly, patterns of 2DG uptake in the normal retina were comparedto that seen in the light-damaged retina, with and without aphotoreceptor transplant. These comparisons were made under twodifferent visual stimulus conditions: 1) darkness and 2) strobe flickerat 10z. FIG. 27 illustrates the results of these comparisons. H&Estained retina with corresponding 2-deoxyglucose autoradiographs. A andB normal retina. Sections cut slightly tangentially to expand retinallayers. C. Dystrophic (light-damaged) retina plus photoreceptortransplant (T) left of arrow. Black and white lines at left on 2DGautoradiograph bracket lower 2DG uptake in inner plexiform and ganglioncell layers. D. Dystrophic retina plus photoreceptor transplant left ofarrow. ONL; outer nuclear layer, T; transplant, DYST; dystrophic,Bar=0.5 mm.

As shown in panel 27A, in darkness 2DG was preferentially taken up inthe outer portion of normal retina (photoreceptors and possibly theinner nuclear layer). As shown in panel 27B, with strobe flickerstimulation 2DG uptake extends through the thickness of the normalretina. These patterns of 2DG uptake are in good agreement with theknown physiological characteristics of the retina.

The outer retina might be expected to show high 2DG uptake in the darksince photoreceptors, horizontal and some bipolar cells are maximallydepolarized in this situation. As strobe flicker is a strong stimulusfor the retina including the amacrine and retinal ganglion cells, 2DGuptake across the entire retina is also to be expected. It thereforeappears that the 2DG uptake pattern in normal retina reflects relativedegrees of neural activity or neural depolarization, and therefore is auseful indicator of neural activity in the retina as it is in otherareas of the nervous system.

In the light-damaged retina which received a photoreceptor transplant,the pattern of 2DG uptake was also dependent on the stimulus conditions.In the dark, preferential uptake of 2DG was limited to the photoreceptortransplant and the adjacent host inner nuclear layer while relativelylower uptake was present in the host's inner plexiform and ganglion celllayers. However, in the strobe flicker condition, high 2DG uptake ispresent in the transplant and, in addition, extended through thethickness of the host's retina--but only in the area of thephotoreceptor graft (FIG. 27D). Adjacent host retina which did notreceive the photoreceptor transplant shows relatively low 2DG uptake.

In darkness, both the normal retina and the light-damaged retinareceiving the photoreceptor transplant show relatively high uptake of2DG in the photoreceptor and inner nuclear layers. The similarity in therelative uptake patterns between these cases suggests that thetransplanted photoreceptors may have similar functional characteristicsas normal photoreceptors (i.e., they depolarize in the dark and arecapable of inducing a sustained depolarization of some cells in thehost's inner nuclear layer).

In strobe flicker, the light-damaged retina receiving the photoreceptortransplant showed high 2DG uptake through the entire thickness of theretina much like that seen in the normal retina under the same stimuluscondition. Adjacent light-damaged retina that did not receive aphotoreceptor transplant showed relatively low 2DG uptake. Thesecomparisons show that the pattern of 2DG uptake in the light-damagedretina approximates that seen in the normal retina only in areas of thehost retina that received photoreceptor grafts. Adjacent areas of thehost retina show relatively low levels of 2DG uptake in both stimulusconditions. The similarity in the 2DG uptake patterns between thelight-damaged retina following photoreceptor grafting and the normalretina in both stimulus conditions suggests that the photoreceptortransplant is capable of light-dependent activation of the light-damagedretina.

EXAMPLE 6

The procedures of Example 1 were repeated except as noted.

While activation of the host retina by the transplanted photoreceptorswas seen with deoxyglucose mapping the nature of this activation wasunclear. Specifically, does such activation represent a nonsynapticmodulation of neurotransmitter release by the transplantedphotoreceptors or are the transplanted cells forming synapses withelements of the host retina?

To address this issue, the ultrastructure of the reconstructed retinawas investigated. Following appropriate survival times, animals wereeuthanized by overdose and immediately enucleated. Control andexperimental eyes for light microscopy were fixed overnight in Bouin'ssolution. Following dehydration and clearing, the tissue was embedded inparaffin. Sections were cut on a rotary microtome. Eyes destined forplastic embedment were fixed for 2 hours in buffered 2.5%glutaraldehyde. After 1/2 hour of aldehyde fixation, the anteriorsegment and lens were removed to facilitate penetration of the fixative.Following primary fixation, eyes for light microscopy were processedfurther for methacrylate embedding. Two to 5 μm sections were cut on arotary microtome using glass "Ralph" knives. In eyes for ultrastructuralanalysis, the area receiving the transplant was localized using the DiIlabel and excess tissue was trimmed before osmium postfixation.Following dehydration and clearing, the tissue was embedded inEpon/Araldite (Mollenhauer, 1964). Blocks were surveyed by stainingsemithin sections with toluidine blue. When the transplant was located,thin sections were cut and stained with uranyl acetate and lead citratefor examination on the EM.

A new outer plexiform-like layer was visible at the interface of thetransplanted ONL and the host inner nuclear layer. Ribbon synapses wereevident within this OPL. These synapses are characteristic of thoseformed by rod photoreceptors, with an electron dense ribbon surroundedby a cluster of vesicles. Ribbon synapses are found only rarely incontrol light-damaged retina. In addition to ribbon synapses, thetransplanted photoreceptors also display inner segments, connectingcilia, and outer segment membranes. These results suggest that synapticconnections between transplanted photoreceptors and host cells were madeindicating that the light-dependent activation may, at least in part, besynaptically mediated.

EXAMPLE 7

The procedures of Example 1 were repeated except as noted.

The functional capabilities of the transplanted photoreceptors andreconstructed retina was ascertained by recording visually evokedcortical potentials ("VEP"). To record the VEP, animals were implantedwith stainless-steel screw electrodes embedded in the skull. The activeelectrodes were placed 2 mm anterior to lambda (bilaterally), andreferred to a second electrode placed anterior to bregma. Bothelectrodes were placed 2 mm lateral to the midline and positioned on thedura. A third screw was placed above the nasal cavity to serve as aground electrode.

Responses of the VEP were elicited by strobe flash test stimuligenerated by a GRASS PS-2 photostimulator directed toward one eye withthe other eye covered by a patch. Responses were differentiallyamplified (GRASS P-15D preamp), displayed on a TECTRONIX #564oscilliscope and then averaged by a MACINTOSH IIx computer usingLABVIEW.

It was found that the reconstructed retina can produce a light-evokedelectrical response in the visual cortex whereas the unreconstructedfellow eye showed little or no response to the same light stimulus.

EXAMPLE 8

The procedures of Example 1 were repeated except as noted.

With indications that neural activity is generated in the centralnervous system by the photoreceptor transplant and the reconstructedretina the question arises at to whether this neural activity can beprocessed appropriately by the central nervous system to produce anappropriate behavioral response to the sensory stimuli. Previous studieshave shown that neural transplants to the brain can restore appropriatebehavioral activity (Bjorklund et al., Neural Graftin g in the MammalianCNS. Elsevier, Amsterdam, 1985). Klassen and Lund Proc. Natl. Acad. Sci.USA 84: 6958-6960, 1987; and Exp. Neurol. 102: 102-108, 1988 have shownthat neural transplants can restore the pupillary reflex mediated byintracranial transplantation of embryonic retinas thus showing thatneural transplants consisting of sensory tissue are capable of mediatinga behaviorally appropriate response to sensory stimulation.

Rats with dystrophic retinas received a photoreceptor transplant asdescribed in Example 1. At various post-surgical time intervals (E.G.,2, 4 and 8 weeks, etc.) animals were anesthesized and held in astereotaxic device. An infrared video camera was focused on the eyethrough an operating microscope and the eye illuminated with infraredlight. To test for pupillary reflex, a light beam controlled by a camerashutter within the operating microscope was used. This light was focusedon the eye. The pupillary response to the light at graded intensities(intensity of the light was controlled by neutral density filters) wasrecorded by video camera connected to a frame grabber system. Thepupillary reflex was then analysed using automated imageprocessingsoftware (ULTIMATE, GTFS, Inc.)

It was found that retinas reconstructed with photoreceptor transplantsdo in fact show a comparatively normal pupillary reflex to light(pupillary constriction) whereas the fellow dystrophic eye shows only aminimal reflex that is aberrant in form (pupillary dilation). Theresults are shown in FIG. 28a, 28b, 28c and 28d. Panels a and b are ofthe reconstructed retina. Panel a shows the iris at light onset whereaspanel b shows the same eye at 5 seconds after light onset. Comparison ofpanel a to panel b shows a normal pupillary constriction mediated bylight. Panels c and d show the fellow blinded eye that received shamsurgery with panel c showing the iris at light onset and panel d showingthe iris 5 seconds later. Comparison of panels c and d show an increasein pupil size with light. This response is aberrant in form and ischaracteristic of individuals suffering from severe retinal dystrophy ofa photoreceptor type.

These results show that neural transplantation can reconstruct thehost's own sensory end organ--in this case the eye--to restore anappropriate behavioral response (i.e., the pupillary reflex) to sensorystimuli. These results have profound significance for the feasibility ofthe restoration of vision by photoreceptor transplantation.

EXAMPLE 9

The procedures of Example 1 were repeated except as noted.

Photoreceptors were taken from mature macaque retina (animals sacrificedfor other research) or the retina of donated human eyes (obtained fromthe St. Louis Eye Bank) using vibratome sectioning of the flat-mountedretina to isolate the intact outer nuclear layer. Hosts were maturemacaque monkeys treated with iodoacetic acid (30 mg/kg given on 3successive days) which selectively eliminates host photoreceptors innon-macular areas of the retina while leaving the remaining retinaintact. This treatment did not compromise central vision and thereforemaintained sight required for behavioral and physiologically importantfunctions (e.g., locating of food and water, visually guided locomoteractivities, grooming, maintenance of circadian rhythms).

The isolated outer nuclear layer was transplanted following a pars planavitrectomy (a standard surgical technique) using a trans-scleralapproach to the subretinal space. The photoreceptors were inserted undera focal retinal detachment induced by the formation of a subretinalbleb. The bleb was created by the infusion of ophthalmic balanced saltsolution. The reconstructed retina was reattached to the back of the eyeby pneumatic tamponade with the transplanted photoreceptors interposedbetween the retina and the underlying pigment epithelium. Dailyinjections of cyclosporin A and dexamethasone were made to suppress anypossible transplant rejection.

It was found human photoreceptors survive transplantation to thenon-human primate eye for as long as tested (2 weeks). These resultsindicate that mature human photoreceptors can be transplanted to thenon-human primate eye. Since the non-human primate eye is almostidentical to the human eye it is expected that human photoreceptors canbe successfully transplanted to the human eye.

From the foregoing description those skilled in the art will appreciatedthat all aspects of the present invention are realized. The presentinvention provides an improved surgical instrument that is adapted toprovide cell organization during transplantation of the photoreceptors.With the surgical instrument of this invention cell organization ismaintained during photoreceptor, RPE, and choroidal transplantationwhile minimizing trauma to the transplanted tissues, the host eye andretina. It is believed that retina reattachment and subsequentsubstantially normal function of the reconstructed retina, in view ofthe transplant, is thereby facilitated. The present invention providesan improved surgical instrument that is constructed to allow relativelylarge expanses of the RPE, choroidea, and photoreceptor cell matrix orcolumn to be transplanted to a sub-retinal space. Maintaining normallayer configuration of the photoreceptors, RPE, and choroidea allowsthese tissues to be transplanted to the appropriate position within theeye. The subsequent integration of the transplanted photoreceptors, RPE,and choroidea with the blinded retina facilitates reconstruction of theblinded retina. The present invention provides an improved surgicalinstrument that allows appropriate retinotopic positioning. The presentinvention provides an improved surgical instrument that protectsphotoreceptors from damage as the surgical device is positioned in theeye. The present invention provides a method of photoreceptor or retinalpigment epithelium isolation and transplantation that, maintains to theextent possible the normal organization of the outer nuclear layer andthese other tissues. The present invention provides a method of cell andtissue isolation by which cells can be isolated without disruption oftheir intercellular organization. With the method of this inventionretinal cells, such as retinal photoreceptors can be isolated withoutthe disruption of the intercellular organization of the outer nuclearlayer or other layer of the retina, RPE, and choroidea.

A number of features of the transplanted cells are that they are andremain alive; they produce opsin, important for phototransduction; theyare functional (i.e., activated by light); and the transplantedphotoreceptors activate a previously blinded retina in a light dependentfashion.

Attachment of retinal tissue to the gelatin substrate allows extendedperiods of in vitro culture of retinal tissues by: maintainingorganization of tissue in culture; and allowing for a better viabilityof cultured tissue.

While a number of embodiments have been shown and described, manyvariations are possible. Photoreceptors can be transplanted to retina inwhich the host's or recipient's photoreceptors are lost by environmental(constant light) or inherited defects. (See: S. E. Hughes and M. S.Silverman (1988) in "Transplantation of retinal photoreceptors todystrophic retina", Soc. Neurosci. Abstr., 18: 1278.) Furthermoretransplanted photoreceptor cells maintain basic characteristics ofnormal photoreceptor cells by producing opsin and maintaining anintercellular organization and apposition to the host retina that issimilar to that seen in the normal outer nuclear layer. The surgicalinstruments may be larger for use in humans. Other approaches to thesubretinal space may be used, e.g., trans-scleral, choroidal. Othersubstrates besides gelatin can be used, e.g. agar, agarose, in factimproved substrates could include factors that can be integrated intogelatin, for example, neurotrophic factors). It is believed thatattachment to gelatin or equivalent substrates will allow prolonged invitro culture, or cryogenic freezing, and similar storage, whileallowing for the maintenance of tissue organization and viability.Finally, it is believed that other methods of attaching retina tosubstrate can be used, such as lectins, or photo-activated cross-linkingagents.

It has been shown that this invention provides a method to isolate theintact photoreceptor layer. This is significant because it will benecessary to maintain tight matrix organization if coherent vision is tobe restored to the retina comprised by the loss of photoreceptors. Asurgical approach has been disclosed which minimizes trauma to the eyeand allows controlled positioning of sheets of transplantedphotoreceptors to their homotopic location within the eye. In additionthese methods for transplantation and isolation of photoreceptors couldbe utilized to prepare and transplant other retinal layers so thatselected populations of retinal cells can be used in otherneurobiological investigations and clinical procedures. It is believedthat these other retinal layers, once they are flattened, appropriatelysectioned, and appropriately affixed to a stabilizing substrate or base,could be prepared for transplantation, storage (e.g., in vitro,cryogenic), or culturing similar to the methods described herein forphotoreceptor layers.

The necessity for prompt re-vascularization typically limits the abilityto transplant most neural tissue, but not photoreceptors. Thephotoreceptor layer of a retina and the ("RPE") is non-vascularized.Non-vascularized tissue shows the least amount of tissue rejection.Consequently, it is believed that genetically dissimilar photoreceptorcells may be transplanted in accordance with the present invention.Matching of host and donor histocompatibility antigens will probably bnecessary for transplantation of the retinal pigment epithelium andchoroidea.

Photoreceptors can be transplanted when developing or when mature. Notonly can mature rat photoreceptors be transplanted, but maturephotoreceptors from human donors can be transplanted as well. This issignificantly different from neurons which must be immature in order tobe transplanted. At present the reason for this difference is not knownbut has obvious importance for retinal and neural transplantationresearch in general.

Finally, transplanted photoreceptors activate the host's or recipient'sdystrophic retina in a light dependent manner that closely resembles theactivation pattern seen in normal retina.

In view of the above, it will be seen that the several objects of theinvention are achieved and other advantages attained.

As various changes could be made in the above surgical instruments,compositions of matter and methods. Without, departing from the scope ofthe invention, it is intended that all matter contained in the abovedescription or shown in the accompanying drawings shall be interpretedas illustrative and not in a limiting sense.

What is claimed:
 1. A mammalian eye tissue graft, comprising mammalianeye tissue isolated from a donor eye laminated to a non-toxic andflexible support, said tissue consisting essentially of the outernuclear layer of a retina, said outer nuclear layer having the samecellular organization as in the donor eye, wherein said support will notinterfere with the viability of said tissue upon transplantation of thegraft into a mammalian eye.
 2. The eye tissue graft of claim 1, whereinsaid outer nuclear layer comprises photoreceptor cells.
 3. The eyetissue graft of claim 1, wherein said support comprises gelatin.
 4. Theeye tissue graft of claim 2, wherein said support comprises gelatin. 5.A method of forming a mammalian eye tissue graft, comprising the stepsof:affixing mammalian eye tissue to a support, said eye tissuecomprising cells, said eye tissue having a first portion and a secondportion, wherein said second portion is affixed to said support;removing said first portion without altering the organization of saidcells forming said second portion so as to form a graft comprising atleast part of said second portion affixed to at least part of saidsupport, said support being non-toxic and flexible, said graft beingcapable of transplantation into a host eye, wherein said first portioncomprises cells found in the inner nuclear layer of a retina, and saidsecond portion comprises photoreceptor cells.
 6. A mammalian eye tissuegraft, comprising mammalian eye tissue isolated from a donor eyelaminated to a non-toxic and flexible support, said eye tissuecomprising at least one layer of cells found in tissue selected from thegroup consisting of the outer nuclear layer of a retina, retinal pigmentepithelium, and choroidal tissue, said cells comprising said isolatedlayer having the same organization as in the donor eye, wherein theinner nuclear layer of the retina is substantially absent from saidgraft, said support will not interfere with the viability of saidisolated tissue upon transplantation of the graft into a host eye, andsaid graft can be transplanted so that said cells will have the samepolarity with respect to the retina in the host eye as said cells had inthe donor eye.
 7. The graft of claim 6, wherein said isolated eye tissuecomprises photoreceptor cells.
 8. The graft of claim 6, wherein saidsupport comprises gelatin.
 9. The graft of claim 7, wherein said supportcomprises gelatin.
 10. A method of forming a mammalian eye tissue graftfor transplantation to a host eye, comprising the steps of:affixingmammalian eye tissue from a donor eye to a support, said eye tissuecomprising cells found in tissue selected from the group consisting ofretinal tissue, retinal pigment epithelium, and choroidal tissue, saideye tissue having a first portion and a second portion, wherein saidsecond portion is affixed to said support; removing said first portionwithout altering the organization of the cells forming said secondportion so as to form a graft comprising at least part of said secondportion affixed to said support, said support being non-toxic andflexible, said graft being capable of transplantation into a host eye,wherein said second portion comprises cells found in tissue selectedfrom the group consisting of the outer nuclear layer of the retina,retinal pigment epithelium, and choroidal tissue, wherein said supportwill not interfere with the viability of said isolated tissue upontransplantation of the graft into a mammalian eye, and said graft can betransplanted to the subretinal space of a host eye so that said cellscomprising said graft will have the same polarity with respect to theretina in the host eye as said cells had in the donor eye.
 11. Themethod of claim 10, wherein said eye tissue comprises retinal tissuehaving an inner nuclear layer and an outer nuclear layer, and said firstportion comprises said inner nuclear layer, and said second portioncomprises said outer nuclear layer.
 12. The method of claim 11, whereinsaid support comprises gelatin, and said second portion comprisesphotoreceptor cells.
 13. A method of forming a mammalian eye tissuegraft and transplanting the graft to a host eye, comprising the stepsof:affixing mammalian eye tissue from a donor eye to a support, said eyetissue comprising cells found in tissue selected from the groupconsisting of retinal tissue, retinal pigment epithelium, and choroidaltissue, said eye tissue having a first portion and a second portion,wherein said second portion is affixed to said support; removing saidfirst portion without altering the organization of the cells formingsaid second portion so as to form a graft comprising at least part ofsaid second portion affixed to said support, said support beingnon-toxic and flexible, said graft being capable of transplantation intoa host eye, wherein said second portion comprises cells found in tissueselected from the group consisting of the outer nuclear layer of theretina, retinal pigment epithelium, and choroidal tissue, wherein saidsupport will not interfere with the viability of said isolated tissueupon transplantation of the graft into a mammalian eye; andtransplanting said graft beneath the retina of a host eye so that saidcells will have the same polarity with respect to the retina in the hosteye as said cells had in the donor eye.
 14. The method of claim 13,wherein said second portion comprises photoreceptor cells.
 15. Themethod of claim 14, wherein said support comprises gelatin.